Why bother to identify Chironomus species used for contaminant monitoring?Isabelle Proulx (isabelle.proulx@ete.inrs.ca) and Landis Hare

Quebec City (Quebec, Canada)

Chironomid larvae have great potential as contaminant biomonitors because they are present in a wide variety of freshwaters, including those that are highly contaminated. Since chironomid species are notoriously difficult to identify, closely related species are often pooled for contaminant analysis. However, in doing so, one presumes that contaminant concentrations do not differ markedly amongthe pooled species. We tested this assumption on the widespread midge Chironomus by collecting larvae of this genus from lakes located along a metal-contamination gradient and then identifying species using a combination of molecular and morphological techniques. By measuring trace metal concentrations in each Chironomus species we discovered that, within a given lake, the concentrationsof some metals (Cd and Se) differed among sympatric species. Among lakes, metal concentrations in the various Chironomus species differed in a consistent manner such that certain species had consistently higher concentrations of some metals than did others. To determine why species sharing the same habitat differ in their metal concentrations, we measured larval sulfur isotopic ratios. Thesemeasurements revealed that Chironomus species sharing the same habitat tend to feed at different depths in sediment where metal concentration and/or bioavailability are likely to differ. Overall, our results suggest that a “one-size fits all” approach for Chironomus species may not be valid and that behavioral differences among these morphologically-similar larvae influence their exposure tocontaminants.

In principle, measurements of contaminants inburrowing chironomid larvae can be used toestimate bioavailable contaminant concentrationsin sediments.

However, before measuring contaminants in chironomids, larvae need to be identified, but ...

Is pooling chironomid species justifiable in contaminant studies?

One way around this constraint is to pool species.

species identification can

be very challenging!!

Introduction

We collected fourth-instar Chironomus larvaefrom a single site in each of 28 lakes in Quebecand Ontario, Canada.

We used the following tools to sort and identify Chironomus larvae to species:

- morphology (mouthparts, tubuli, etc.),- number and structural arrangement of giant salivary chromosomes,

- RFLP analyses,- DNA barcoding using a mitochondrial gene:

cytochrome C oxidase subunit I (COI).

Differences among Chironomus species in the colour of the gut contents led us to believe that their feeding habits differed. To test this idea, we measured stable sulphur isotopic ratios ( 34S) in Chironomus larvae, where

δ34S = [(34Ssample/32Ssample)/(34Sstandard/32Sstandard )-1] * 1000

This approach is based on the fact that animals feeding on oxic particles tend to have higher 34S values than do those feeding on anoxic particles (Martin et al. 2008; Croisetière et al. 2009)

Figure 1. Neighbour-joining tree based on COI sequences of Chironomus species collected from 28 lakes (larvalmorpho-type and lake names are indicated) and of species sequences obtained from either the Barcode of LifeData Systems (labelled as BOLD with the species name and the accession number) or from GenBank (labelled asGenBank with the species name and the accession number). C. = Chironomus, G. = Glyptotendipes, D. = Drosophila

Figure 2. Mean (± SD) cadmium (Cd) (A) and selenium (Se) (B) concentrations invarious Chironomus species (see symbols in figure 1) collected from 5 lakes (asmeasured by ICP-MS). In a given lake, values that do not differ significantly (p >0.05) are followed by the same letter.

Collection of larvae

We tested the validity of this practice bymeasuring trace elements in various species ofChironomus collected from lakes situated along acontamination gradient in northern Canada.

Abstract

Results

Figure 3. Mean 34S values (‰, ±SD) for various Chironomus species (seesymbols in figure 1) collected from 5 lakes. In a given lake, values that donot differ significantly (p > 0.05) are followed by the same letter.

Croisetière L., Hare L., Tessier A. & Cabana G. (2009) Sulphur stable isotopes candistinguish trophic dependence on sediments and plankton in boreal lakes.Freshwater Biology 54: 1006–1015.

Martin J. (2010, with updates) North American cytospecies of the genusChironomus (includes Camptochironomus, Chaetolabis, Lobochironomusand some Einfeldia). Available from: http://www.genetics.unimelb.edu.au/Martin/NACytfiles/NAChiron.html (9 March 2010).

Martin S., Proulx I. & Hare L. (2008) Explaining metal concentrations insympatric Chironomus species. Limnology and Oceanography 53: 411–419.

References

- Feeding behaviours can differ among sympatric Chironomusspecies.

- Behavioural differences among species influence their exposure tocontaminants.

- A “one-size fits all” approach may not be valid for taxa such asChironomus.

- Species differences should not be disregarded in risk assessments.

Conclusions

Colour of gut contents

34S → more +

34S → more -Cd less bioavailable

Se more bioavailable

Cd more bioavailableSe less bioavailable

-[Cd] and [Se] can differ widely among Chironomus species.

-Within a given lake, some species always have higher concentrations than do others.

-For Cd, the species represented by the circles ( ) always have higher concentrations than do the other Chironomus species ( ).

-For Se, the reverse is true.

-The same pattern was observed in all 28 lakes!

-Within a given lake, some species ( ) consistently have more positive 34S values than do others ( ).-Species having higher 34S values ( ) feed on oxic sediments, where Cd is more bioavailable and Se is less bioavailable. The reverse is the case for species having lower 34S values ( ).-The trend is the same in all 28 lakes!

Chironomus species feedingon oxic sediments

Chironomus species feeding on anoxic sediments

to be determined…

C. entisC. staegeri

C. (Camptochironomus) dilutus

C. cuciniC. harpi

C. major (?) C. tigris

unidentified species #1unidentified species #2unidentified species #3unidentified species #4unidentified species #5unidentified species #6

C. sp. 2g (Martin 2010)C. maturus

Table 1. Feeding behaviours of Chironomus species as deduced from 34S measurements in larvae.

Why do Chironomus species sharing the same habitat differ in their trace element concentrations?

Acknowledgements

Special thanks to MelissaCarew, John Martin and theUniversity of Melbourne(Australia) for hosting IsabelleProulx.

semireductus to plumosus type- Kinojevis

semireductus to plumosus type- Osisko(1)

semi... to plumosus type - Alembert(2)

semireductus to plumosus type - Duprat

semi... to plumosus type - Marlon(2)

semireductus to plumosus type - Dasserat

semireductus to plumosus type- Duprat(2)

semi... to plumosus type - Pelletier(3)

semi... to plumosus type - Marlon(3)

semi... to plumosus type - Pelletier(2)

semi... to plumosus type - Alembert(3)

semireductus to plumosus type- Opasatica

semireductus to plumosus type - Augustin

semireductus to plumosus type - Kelly

semireductus to plumosus type - Rouyn

semireductus to plumosus type- Duprat(1)

semi... to plumosus type - Pelletier(4)

semi... to plumosus type - Pelletier(1)

semi... to plumosus type - Marlon(1)

GenBank - C.entis(AF192195.1)

semireductus to plumosus type- Osisko(2)

semireductus to plumosus type - Marlon

plumosus type - Bibby

plumosus type - Mc Farlane

plumosus type - Tilton

plumosys type - St-Joseph

plumosus type - Silver 2

plumosus type - Kinojevis

plumosus type - St-Joseph

plumosus type - Alembert

plumosus type - Opasatica

BOLD - C.staegeri(COTW045-11|UMA.5.111M)

plumosus type - Military Base

plumosus type - Silver

plumosus type - Crooked

plumosus type - Duprat

thummi type - Bibby

thummi type - Arnoux

thummi type - Pine

thummi type - Raft

thummi type - Osisko

thummi type - Hannah

thummi type - Ramsey

bathophilus type - Kinojevis

bathophilus type - Mc Farlane

bathophilus type - St-Joseph

bathophilus type - Alembert

bathophilus type - Opasatica

bathophilus-type - Arnoux

bathophilus type - Adeline

bathophilus type - Saint-Charles

GenBank - C. sp. 2g.(DQ648205.1)

salanarius type - Clearwater

salanarius type - Raft

salanarius type - Opasatica

salanarius type - Vaudray

plumosus type - Arnoux 8

BOLD - C.harpi(COTW024-08|UIL.1.1 F)

bathophilus type - Silver

GenBank - C.maturus(DQ648204.1)

plumosus type - Bedard

salanarius type - Mc Farlane

salanarius type - Alembert

salanarius type - Ramsey

salanarius type - Hannah

plumosus type - Kelly

plumosus type - Military

GenBank - C.dilutus(AF110162.1)

thummi type - Marlon

BOLD - G.lobiferus(GBDP4109-07|DQ648213)

BOLD - D.affinis (TDWGB659-10|TDWG-0795)

C. entis(possibility of 2 species : C. entis and C. plumosus)

C. tigris

C. staegeri

unidentified species #1

unidentified species #2

unidentified species #3

unidentified species #4

C. sp. 2g (Martin 2010)

C. cucini

C. harpiunidentified species #5

C. maturus

C. major (?)

C. (Camptochironomus) dilutus

unidentified species #6

Do trace element concentrations differ between sympatric Chironomus species?